Therapeutic compounds and methods of use

ABSTRACT

The invention relates to protein binding interacting/binding compounds and methods of identifying and using them. The invention further relates to pharmaceutical compositions and methods for treating 5-HT2C disorders, including diseases and disorders mediated by GPCRs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application No.Ser. 15/864,562, filed Jan 8, 2018, which is a continuation applicationof U.S. application Ser. No. 14/861,826, filed Sep. 22, 2015, now issuedU.S. Pat. No. 9,862,674; which is a continuation application of U.S.application Ser. No. 12/452,108, filed Apr. 19, 2010, abandoned; whichis the national stage application pursuant to 35 U.S.C. § 371 of PCTapplication PCT/US2008/007457, filed Jun. 13, 2008; which claims thebenefit of U.S. Provisional Patent Application No. 60/934,689, filedJun. 15, 2007, the contents of which are incorporated herein byreference in their entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with government support under Grant No. MH068655awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Serotonin (5-hydroxytryptamine, 5HT) mediates a wide variety of centraland peripheral psychological and physiological effects through 14mammalian 5HT receptor subtypes that are grouped into the 5HT₁-5HT₇families (Sanders-Bush and Mayer, 2006). The 5HT2 family consists of the5HT_(2A), 5HT_(2B), and 5HT_(2C) membrane-bound G protein-coupledreceptors (GPCRs) that signal primarily through Gα_(q) to activatephospholipase (PL) C and formation of inositol phosphates (IP) anddiacylglycerol (DAG) second messengers (Raymond et al., 2001). The human5HT_(2C) receptor (Saltzman et al., 1991) apparently is foundexclusively in the brain where it is widely expressed and putativelyinvolved in several (patho)-physiological and psychological processes,including, ingestive behavior (Tecott et al., 1995), cocaine addiction(Fletcher et al., 2002; Rocha et al., 2002; Muller and Huston, 2006),sleep homeostasis (Frank et al., 2002), anxiety (Kennett et al., 1994;Sard et al., 2005; Heisler et al., 2007), depression (Tohda et al.,1989; Palvimaki et al., 1996), epilepsy (Heisler et al., 1998),Alzheimer's disease (Arjona et al., 2002; Stein et al., 2004), motorfunction (Heisler and Tecott, 2000; Segman et al., 2000), psychosis(Marquis et al., 2007; Siuciak et al., 2007) and response toantipsychotic drugs (Veenstra-VanderWeele et al., 2000; Reynolds et al.,2005). Thus, the importance of the 5HT_(2C) receptor as apharmacotherapeutic target has been apparent for about 10 years,however, no 5HT_(2C)-specific drugs have been developed.

One challenge regarding drug discovery targeting the 5HT_(2C) receptoris that this GPCR shares a transmembrane domain (TMD) sequence identityof about 80% with the 5HT_(2A) receptor and about 70% with the 5HT_(2B)receptor (Julius et al., 1988; 1990). The highly conserved TMDs andsimilar second messenger coupling has made development of agonistligands selective for the 5HT_(2C) receptor especially difficult.Nevertheless, there is compelling evidence that activation of 5HT_(2C)receptors reduces food intake and leads to anti-obesity effects. Forexample, 5HT_(2C) knockout mice demonstrate increased feeding andobesity, and, they are resistant to the anorectic effects ofd-fenfluramine (Tecott et al., 1995; Vickers et al., 1999; 2001; Heisleret al., 2002). Fenfluramine now is banned, because, although peopleusing the drug showed weight loss due to activation of brain 5HT_(2C)receptors, fenfluramine also activates 5HT_(2A) receptors that may leadto adverse psychiatric (hallucinogenic) effects (Nichols, 2004) and5HT_(2B) receptors which causes valvular heart disease (Connolly et al.,1997; Fitzgerald et al., 2000; Rothman et al., 2000; Roth, 2007) andpulmonary hypertension (Pouwels et al., 1990; Launay et al.,2002)—fatalities have resulted from the 5HT_(2B)-mediated effects.

Although an agonist ligand truly selective for 5HT_(2C) vs. 5HT_(2A)and/or 5HT_(2B) receptors has not been reported until this paper, it hasbeen possible to partially elucidate the role of brain 5HT_(2C)receptors to attenuate cocaine use and dependence using very selective(i.e., at least 100-fold) 5HT_(2A) and 5HT_(2C) antagonists in ratcocaine self-administration paradigms. For example, the selective5HT_(2A) antagonist M100907 (Kehne et al., 1996) does not alterresponding rate for cocaine self-administration but the selective5HT_(2C) antagonist SB242084 (Bromidge et al., 1997) increases the rateof cocaine self-administration dose-dependently (Fletcher et al., 2002).The tremendous potential of 5HT_(2C) agonist pharmacotherapy forpsychostimulant addiction now is widely recognized (Bubar andCunningham, 2006).

The pharmacotherapeutic relevance of the 5HT_(2C) receptor in obesityand neuropsychiatric disorders such as psychostimulant addiction hasstimulated intense interest by pharmaceutical companies to develop aselective 5HT_(2C) agonist, however, all 5HT_(2C) agonists reported sofar also activate 5HT_(2A) and/or 5HT_(2B) receptors (Nilsson, 2006).Nevertheless, the 5HT₂ agonist lorcaserin (APD356) recently went toPhase III clinical trials for obesity treatment even though it has onlya modest 15-fold selectivity for activation of 5HT_(2C) receptors over5HT_(2A) receptors (Jensen, 2006; Smith et al., 2006).

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a neuropsychiatric disorder comprisingadministering to the subject in need thereof a therapeutically effectiveamount of a compound capable of modulating GPCR binding interactions. Inone embodiment, the compound is capable of agonizing a 5HT2c. In anotherembodiment, the compound is capable agonizing a 5-HT2c, whileantagonizing 5-HT2a and/or 5-HT2b.

In one aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a neuropsychiatric disorder. The methodincludes administering to a subject in need thereof a therapeuticallyeffective amount of a GPCR modulating compound.

In another aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a neuropsychiatric disorder. The methodincludes administering to a subject in need thereof a therapeuticallyeffective amount of a compound capable of modulating 5-HT2 bindinginteractions by directly modulating 5-HT2c, preferably selectivelyrelative to 5-HT2a and/or 5-HT2b.

In another embodiment, the invention provides a method of treating asubject suffering from or susceptible to a neuropsychiatric disorder.The method includes administering to a subject identified as in needthereof a therapeutically effective amount of a 5-HT2c agonizingcompound or a 5-HT2c selective compound.

In another aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a neuropsychiatric disorder, includingobesity, addiction, cocaine addiction, psychosis, anxiety, sleephomeostasis and the like. The method includes administering to a subjectagonizing 5-HT2c.

In another aspect, the invention provides a method of treating a subjectsuffering from or susceptible to obesity, addiction, cocaine addiction,psychosis, anxiety, or sleep homeostasis, comprising administering tothe subject an effective amount of a compound capable of agonizing5-HT2c (including selectively relative to 5-HT2a and/or 5-HT2b), suchthat the subject is treated.

In one aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a GPCR disorder comprisingadministering to subject in need thereof a therapeutically effectiveamount of a compound capable of modulating GPCR binding interactions. Inone embodiment, the compound is capable of agonizing a GPCR. In anotherembodiment, the compound is capable antagonizing a GPCR.

In one aspect, the invention provides a method of treating or preventinga GPCR-mediated disorder in a subject comprising administering to thesubject identified as in need thereof a APT compound. In certainembodiments, the APT compound is a compound of Table 1 (infra). Incertain embodiments, the APT compound is:

wherein,

R₁ is independently H or optionally substituted phenyl (e.g., halo,alkoxy, CF₃); and

R₂ is independently H or optionally substituted phenyl (e.g., halo,alkoxy, CF₃); or salt, hydrate or solvate thereof.

In certain embodiments, in the APT compound only one of R₁ and R₂ is H.In certain embodiments, in the APT compound only one of R₁ and R₂ isoptionally substituted phenyl.

In certain embodiments, the disorder is a neuropsychiatric disorder(e.g., obesity, addiction, anxiety, depression, schizophrenia, and sleepdisorders), a neurodegenerative disorder (e.g., Parkinson's Disease,Alzheimer's Disease), a neurological disorder (e.g., epilepsy), acardiovascular disorder (e.g., hypertension), a gastrointestinaldisorder (e.g., irritable bowel syndrome), or a genitor-urinary tractdisorder (e.g., bladder control). In certain embodiments, the disorderis cocaine addiction. In certain embodiments, the disorder is obesity.

In one aspect, the invention provides a method of inhibiting 5-HT2C in asubject identified as in need of such treatment, comprisingadministering a APT compound.

In another aspect, the invention provides a method of treating obesityin a subject comprising administering to the subject identified as inneed thereof a APT compound capable of selectively inhibiting the 5-HT2crelative to 5-HT2a or 5-HT2b. In certain embodiments, the bindinginteraction for inhibiting 5-HT2c is at least 5-fold (alternatively atleast 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 500 fold) greaterthan for either 5-HT2a or 5-HT2b. In certain embodiments, the bindinginteraction for inhibiting 5-HT2c is at least 100-fold greater than foreither 5-HT2a or 5-HT2b.

In another aspect, the invention provides a method for identifying acompound that is capable of modulating 5-HT2c activity comprising; (i)producing a three-dimensional representation of a molecule or molecularcomplex, wherein said molecule or molecular complex comprises a bindingpocket defined by structure coordinates of 5-HT2c; or b) athree-dimensional representation of a homologue of said molecule ormolecular complex, wherein said homologue comprises a binding pocketthat has a root mean square deviation from the backbone atoms of saidamino acids of not more than about 2.0 angstroms; (ii) producing athree-dimensional representation of a test compound; (iii) assessing thebinding interaction of the test compound with the target. In certainembodiments, the method further comprises contacting the test compoundwith a 5-HT2c and measuring the binding activity of the compound.

In another aspect, the invention provides a method for identifying acompound that modulates 5-HT2c, the method comprising obtaining acrystal structure of a 5-HT2c protein or obtaining information relatingto the crystal structure of a 5-HT2c protein and modeling a testcompound into or on the 5-HT2c protein structure to determine whetherthe compound modulates the interaction of a 5-HT2c protein. In certainembodiments, the step of modeling comprises modeling or determining theability of the compound to bind to or associate with a binding pocketdefined by structure coordinates of the one or more of transmembranedomains 1-7of 5-HT2c.

Yet another aspect of the invention is a method for identifying acompound useful to treat or prevent obesity, addiction, cocaineaddiction, psychosis, anxiety, or sleep homeostasis. The method includescontacting a 5-HT2c complex with a test compound, and evaluating theability of the test compound to modulate (e.g., agonize or antagonize),5-HT2c.

Yet another aspect of the invention is a method for identifying acompound that modulates the activity of 5-HT2c, the method comprisingusing the atomic coordinates of the one or more of transmembrane domains1-7 of 5-HT2c, to generate a three-dimensional structure (e.g., insilico) of a molecule comprising a binding pocket, and employing thethree-dimensional structure to identify a compound that modulates theactivity of the one or more of transmembrane domains 1-7 of 5-HT2c.

In another aspect, the invention provides a compound represented by theformula (I):

wherein,

R₁ is independently H or optionally substituted phenyl (e.g., halo,alkoxy, CF₃); and

R₂ is independently H or optionally substituted phenyl (e.g., halo,alkoxy, CF₃); or salt, hydrate or solvate thereof. In certainembodiments, R₁ and R₂ are not simultaneously the same.

In another aspect, the invention provides a composition comprising acompound of the invention (e.g., a compound of formula (I)) and apharmaceutically acceptable carrier.

In another aspect, the invention provides a method of making acomposition of the invention, the method comprising combining a compoundof the invention (e.g., a compound of formula (I)) and apharmaceutically acceptable carrier.

In another aspect, the invention provides a packaged compositionincluding a therapeutically effective amount of a 5-HT2c agonistcompound (e.g., a compound of formula (I)) and a pharmaceuticallyacceptable carrier or diluent. The composition may be formulated fortreating a subject suffering from or susceptible to a neuropsychiatricdisorder (e.g., obesity), and packaged with instructions to treat asubject suffering from or susceptible to a neuropsychiatric disorder.

In one aspect, the invention provides a kit for treating aneuropsychiatric disorder in a subject is provided and includes acompound herein (e.g., a compound of formula (I)), pharmaceuticallyacceptable esters, salts, and prodrugs thereof, and instructions foruse. In further aspects, the invention provides kits for agonizing5-HT2c, assessing the efficacy of an anti-obesity treatment in asubject, monitoring the progress of a subject being treated with a5-HT2c agonist , selecting a subject with a neuropsychiatric disorderfor treatment with 5-HT2c agonist, and/or treating a subject sufferingfrom or susceptible to a neuropsychiatric disorder (e.g., obesity). Incertain embodiments, the invention provides: a kit for treating aneuropsychiatric disorder in a subject, the kit comprising a compoundcapable of modulating (e.g., agonizing) 5-HT2c agonist activity. Inother aspects the compound selectively agonizes 5-HT2c relative to5-HT2a and/or 5-HT2b. In other aspects the compound selectivelyantagonizes 5-HT2a and/or 5-HT2b.

In another aspect, the invention relates to a three-dimensionalstructure of a one or more of transmembrane domains 1-7 of 5-HT2c, eachalone or combinations thereof.

Thus, the present invention provides molecules or molecular complexesthat comprise either one or both of these binding pockets or homologuesof either binding pocket that have similar three-dimensional shapes.

The invention also provides a pharmaceutical compositions of thecompounds described herein, comprising a compound capable of agonizing5-HT2c; a compound capable of agonizing 5-HT2c selectively relative to5-HT2a and/or 5-HT2b; a compound capable of agonizing 5-HT2c andantagonizing 5-HT2a and/or 5-HT2b; or a pharmaceutically acceptableester, salt, or prodrug thereof, together with a pharmaceuticallyacceptable carrier.

In another aspect, the invention provides a machine readable storagemedium which comprises the structural coordinates of a binding pocketdefining the one or more of transmembrane domains 1-7 of 5-HT2c.

In another aspect, the invention provides a computer for producing athree-dimensional representation of a molecule or molecular complex,wherein said molecule or molecular complex comprises a binding pocketdefined by structure coordinates of the one or more of transmembranedomains 1-7 of 5-HT2c; or b) a three-dimensional representation of ahomologue of said molecule or molecular complex, wherein said homologuecomprises a binding pocket that has a root mean square deviation fromthe backbone atoms of said amino acids of not more than about 2.0angstroms. The computer includes: (i) a machine-readable data storagemedium comprising a data storage material encoded with machine-readabledata, wherein said data comprises the structure coordinates of the oneor more of transmembrane domains 1-7 of 5-HT2c; (ii) a working memoryfor storing instructions for processing said machine-readable data;(iii) a central-processing unit coupled to said working memory and tosaid machine-readable data storage medium for processing said machinereadable data into said three-dimensional representation; and (iv) adisplay coupled to said central-processing unit for displaying saidthree-dimensional representation.

The invention also provides methods for designing, evaluating andidentifying compounds which bind to the aforementioned binding pockets.Other embodiments of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further described below with reference to thefollowing non-limiting examples and with reference to the followingfigures, in which:

FIG. 1. depicts 5- and 6-APT compound structures.

FIGS. 2A-2C depict representative binding curves for [³H]-ketanserinlabeled 5HT_(2A) receptors and [³H]-mesulergine labeled 5-HT_(2B)receptors and 5HT_(2C) receptors.

FIG. 3. depicts representative 5-HT_(2A), 5HT_(2B), and 5HT_(2C)radioligand displacement curves for (-)-trans-PAT.

FIG. 4. depicts assessment of (-)-trans-PAT agonist activity at5HT₂-subtype receptors.

FIGS. 5A-5B depict assessment of the ability of (-)-trans-PAT to act asa 5HT_(2A) and 5HT_(2B) receptor antagonist regarding 5-HT-mediatedstimulation of PLC/[³H]-IP formation.

FIG. 6. depicts curves for 5-HT_(2c) binding.

FIG. 7. depicts curves for 5-HT_(2A) and 5HT_(2C) binding.

FIG. 8. depicts curves for inositol phosphates (IP) activity.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have now discovered a therapeutic strategy thataddresses selective disease treatment and prevention (i.e., havingreduced or minimized adverse side effects) by selectively targeting5-HT2c. Such interactions are relevant for modulation of 5-HT2c mediateddisorders, particularly in certain neuropsychiatric disorder types where5-HT2 mechanisms play a significant role.

The present invention relates, at least in part, to the discovery thatthe 5-HT2c interactions are useful as targets (e.g., selective) forneuropsychiatric disorder therapy.

1. Definitions

Before further description of the present invention, and in order thatthe invention may be more readily understood, certain terms are firstdefined and collected here for convenience.

The term “administration” or “administering” includes routes ofintroducing the compound of the invention(s) to a subject to performtheir intended function. Examples of routes of administration that maybe used include injection (subcutaneous, intravenous, parenterally,intraperitoneally, intrathecal), oral, inhalation, rectal andtransdermal. The pharmaceutical preparations may be given by formssuitable for each administration route. For example, these preparationsare administered in tablets or capsule form, by injection, inhalation,eye lotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administration is preferred. The injection can bebolus or can be continuous infusion. Depending on the route ofadministration, the compound of the invention can be coated with ordisposed in a selected material to protect it from natural conditionswhich may detrimentally effect its ability to perform its intendedfunction. The compound of the invention can be administered alone, or inconjunction with either another agent as described above or with apharmaceutically-acceptable carrier, or both. The compound of theinvention can be administered prior to the administration of the otheragent, simultaneously with the agent, or after the administration of theagent. Furthermore, the compound of the invention can also beadministered in a pro-drug form which is converted into its activemetabolite, or more active metabolite in vivo.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. The term alkyl further includesalkyl groups, which can further include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms. Inpreferred embodiments, a straight chain or branched chain alkyl has 30or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain,C₃-C₃₀ for branched chain), preferably 26 or fewer, and more preferably20 or fewer, and still more preferably 4 or fewer. Likewise, preferredcycloalkyls have from 3-10 carbon atoms in their ring structure, andmore preferably have 3, 4, 5, 6 or 7 carbons in the ring structure.

Moreover, the term alkyl as used throughout the specification andsentences is intended to include both “unsubstituted alkyls” and“substituted alkyls,” the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. Cycloalkyls can be further substituted, e.g., with thesubstituents described above. An “alkylaryl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl”also includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond respectively.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six, and still morepreferably from one to four carbon atoms in its backbone structure,which may be straight or branched-chain. Examples of lower alkyl groupsinclude methyl, ethyl, n-propyl, i-propyl, tert-butyl, hexyl, heptyl,octyl and so forth. In preferred embodiment, the term “lower alkyl”includes a straight chain alkyl having 4 or fewer carbon atoms in itsbackbone, e.g., C1-C4 alkyl.

The terms “alkoxyalkyl,” “polyaminoalkyl” and “thioalkoxyalkyl” refer toalkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond,respectively. For example, the invention contemplates cyano andpropargyl groups.

The term “aryl” as used herein, refers to the radical of aryl groups,including 5- and 6-membered single-ring aromatic groups that may includefrom zero to four heteroatoms, for example, benzene, pyrrole, furan,thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Aryl groups also include polycyclic fused aromatic groups such asnaphthyl, quinolyl, indolyl, and the like. Those aryl groups havingheteroatoms in the ring structure may also be referred to as “arylheterocycles,” “heteroaryls” or “heteroaromatics.” The aromatic ring canbe substituted at one or more ring positions with such substituents asdescribed above, as for example, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato,cyano, amino (including alkyl amino, dialkylamino, arylamino,diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Arylgroups can also be fused or bridged with alicyclic or heterocyclic ringswhich are not aromatic so as to form a polycycle (e.g., tetralin).

The term “associating with” refers to a condition of proximity between achemical entity or compound, or portions thereof, and a binding pocketor binding site on a protein. The association may be non-covalent(wherein the juxtaposition is energetically favored by hydrogen bondingor van der Waals or electrostatic interactions) or it may be covalent.

The term “binding pocket”, as used herein, refers to a region of amolecule or molecular complex, that, as a result of its shape, favorablyassociates with another chemical entity or compound.

The language “biological activities” of a compound of the inventionincludes all activities elicited by compound of the inventions in aresponsive cell. It includes genomic and non-genomic activities elicitedby these compounds.

“Biological composition” or “biological sample” refers to a compositioncontaining or derived from cells or biopolymers. Cell-containingcompositions include, for example, mammalian blood, red cellconcentrates, platelet concentrates, leukocyte concentrates, blood cellproteins, blood plasma, platelet-rich plasma, a plasma concentrate, aprecipitate from any fractionation of the plasma, a supernatant from anyfractionation of the plasma, blood plasma protein fractions, purified orpartially purified blood proteins or other components, serum, semen,mammalian colostrum, milk, saliva, placental extracts, acryoprecipitate, a cryosupernatant, a cell lysate, mammalian cellculture or culture medium, products of fermentation, ascites fluid,proteins induced in blood cells, and products produced in cell cultureby normal or transformed cells (e.g., via recombinant DNA or monoclonalantibody technology). Biological compositions can be cell-free. In apreferred embodiment, a suitable biological composition or biologicalsample is a red blood cell suspension. In some embodiments, the bloodcell suspension includes mammalian blood cells. Preferably, the bloodcells are obtained from a human, a non-human primate, a dog, a cat, ahorse, a cow, a goat, a sheep or a pig. In preferred embodiments, theblood cell suspension includes red blood cells and/or platelets and/orleukocytes and/or bone marrow cells.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “diastereomers” refers to stereoisomers with two or morecenters of dissymmetry and whose molecules are not mirror images of oneanother.

The term “effective amount” includes an amount effective, at dosages andfor periods of time necessary, to achieve the desired result, e.g.,sufficient to treat a disorder delineated herein. An effective amount ofcompound of the invention may vary according to factors such as thedisease state, age, and weight of the subject, and the ability of thecompound of the invention to elicit a desired response in the subject.Dosage regimens may be adjusted to provide the optimum therapeuticresponse. An effective amount is also one in which any toxic ordetrimental effects (e.g., side effects) of the compound of theinvention are outweighed by the therapeutically beneficial effects.

A therapeutically effective amount of compound of the invention (i.e.,an effective dosage) may range from about 0.001 to 30 mg/kg body weight,preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. Theskilled artisan will appreciate that certain factors may influence thedosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a compound of the invention can include a singletreatment or, preferably, can include a series of treatments. In oneexample, a subject is treated with a compound of the invention in therange of between about 0.1 to 20 mg/kg body weight, one time per weekfor between about 1 to 10 weeks, preferably between 2 to 8 weeks, morepreferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. It will also be appreciated that the effectivedosage of a compound of the invention used for treatment may increase ordecrease over the course of a particular treatment.

The term “enantiomers” refers to two stereoisomers of a compound whichare non-superimposable mirror images of one another. An equimolarmixture of two enantiomers is called a “racemic mixture” or a“racemate.”

The term “haloalkyl” is intended to include alkyl groups as definedabove that are mono-, di- or polysubstituted by halogen, e.g.,fluoromethyl and trifluoromethyl.

The term “halogen” designates —F, —Cl, —Br or —I.

The term “hydroxyl” means —OH.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,sulfur and phosphorus.

The term “homeostasis” is art-recognized to mean maintenance of static,or constant, conditions in an internal environment.

The language “improved biological properties” refers to any activityinherent in a compound of the invention that enhances its effectivenessin vivo. In a preferred embodiment, this term refers to any qualitativeor quantitative improved therapeutic property of a compound of theinvention, such as reduced toxicity.

The term “GPCR disorder” includes any disease, disorder or symptomsthereof that are mediated by a G protein-coupled receptor (e.g., 5-HT2a,5-HT2b, 5-HT 2c, muscarinic M1-M5). Diseases and disorders mediated bysuch GPCRs include, for example, neuropsychiatric disorders (e.g.,obesity, addiction, cocaine addiction, psychosis, anxiety, depression,schizophrenia, psychosis, and sleep disorders), neurodegenerativedisorders (e.g., Parkinson's Disease, Alzheimer's Disease), neurologicaldisorders (e.g., epilepsy), cardiovascular disorders (e.g.,hypertension), gastrointestinal disorders (e.g., irritable bowelsyndrome), and genitor-urinary tract disorders (e.g., bladder control).

-   -   The language “M1-M5 GPCR” refers to the cholinergic muscarinic        M1-M5 neurotransmitter G protein-coupled receptors (including        those delineated herein).

The language “5-HT2” refers to the serotonin receptors (including thosedelineated herein) such as 5-HT2a, 5-HT2b and 5-HT2c sub-types.

The term “optionally substituted” is intended to encompass groups thatare unsubstituted or are substituted by other than hydrogen at one ormore available positions, typically 1, 2, 3, 4 or 5 positions, by one ormore suitable groups (which may be the same or different). Such optionalsubstituents include, for example, hydroxy, halogen, cyano, nitro,C₁-C₈alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl, C₁-C₈alkoxy, C₂-C₈alkyl ether,C₃-C₈alkanone, C₁-C₈alkylthio, amino, mono- or di-(C₁-C₈alkyl)amino,haloC₁-C₈alkyl, haloC₁-C₈alkoxy, C₁-C₈alkanoyl, C₂-C₈alkanoyloxy,C₁-C₈alkoxycarbonyl, —COOH, —CONH₂, mono- ordi-(C₁-C₈alkyl)aminocarbonyl, —SO₂NH₂, and/or mono ordi(C₁-C₈alkyl)sulfonamido, as well as carbocyclic and heterocyclicgroups. Optional substitution is also indicated by the phrase“substituted with from 0 to X substituents,” where X is the maximumnumber of possible substituents. Certain optionally substituted groupsare substituted with from 0 to 2, 3 or 4 independently selectedsubstituents (i.e., are unsubstituted or substituted with up to therecited maximum number of substituents).

The term “isomers” or “stereoisomers” refers to compounds which haveidentical chemical constitution, but differ with regard to thearrangement of the atoms or groups in space.

The term “modulate” refers to an increase or decrease, e.g., in theability of a compound inhibit activity of a target in response toexposure to a compound of the invention, including for example in ansubject (e.g., animal, human) such that a desired end result isachieved, e.g., a therapeutic result.

The term “obtaining” as in “obtaining a compound capable of modulating(agonizing, antagonizing) a target delineated herein and is intended toinclude purchasing, synthesizing or otherwise acquiring the compound.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The terms “polycyclyl” or “polycyclic radical” refer to the radical oftwo or more cyclic rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbonsare common to two adjoining rings, e.g., the rings are “fused rings”.Rings that are joined through non-adjacent atoms are termed “bridged”rings. Each of the rings of the polycycle can be substituted with suchsubstituents as described above, as for example, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “prodrug” or “pro-drug” includes compounds with moieties thatcan be metabolized in vivo. Generally, the prodrugs are metabolized invivo by esterases or by other mechanisms to active drugs. Examples ofprodrugs and their uses are well known in the art (See, e.g., Berge etal. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19). The prodrugscan be prepared in situ during the final isolation and purification ofthe compounds, or by separately reacting the purified compound in itsfree acid form or hydroxyl with a suitable esterifying agent. Hydroxylgroups can be converted into esters via treatment with a carboxylicacid. Examples of prodrug moieties include substituted andunsubstituted, branch or unbranched lower alkyl ester moieties, (e.g.,propionoic acid esters), lower alkenyl esters, di-lower alkyl-aminolower-alkyl esters (e.g., dimethylaminoethyl ester), acylamino loweralkyl esters (e.g., acetyloxymethyl ester), acyloxy lower alkyl esters(e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-loweralkyl esters (e.g., benzyl ester), substituted (e.g., with methyl, halo,or methoxy substituents) aryl and aryl-lower alkyl esters, amides,lower-alkyl amides, di-lower alkyl amides, and hydroxy amides. Preferredprodrug moieties are propionoic acid esters and acyl esters. Prodrugswhich are converted to active forms through other mechanisms in vivo arealso included.

The language “a prophylactically effective amount” of a compound refersto an amount of a compound of the invention any formula herein orotherwise described herein which is effective, upon single or multipledose administration to the patient, in preventing or treating a disorderherein.

The language “reduced toxicity” is intended to include a reduction inany undesired side effect elicited by a compound of the invention whenadministered in vivo.

The term “sulfhydryl” or “thiol” means —SH.

The term “subject” includes organisms which are capable of sufferingfrom a disorder herein or who could otherwise benefit from theadministration of a compound of the invention, such as human andnon-human animals. Preferred humans include human patients sufferingfrom or prone to suffering from a neuropsychiatric disorder orassociated state, as described herein. The term “non-human animals” ofthe invention includes all vertebrates, e.g., mammals, e.g., rodents,e.g., mice, and non-mammals, such as non-human primates, e.g., sheep,dog, cow, chickens, amphibians, reptiles, etc.

The term “susceptible to a neuropsychiatric disorder” is meant toinclude subjects at risk of developing a neuropsychiatric disorder,e.g., including those delineated herein, i.e., subjects suffering from aneuropsychiatric disorder or symptom thereof, subjects having a familyor medical history of neuropsychiatric disorder or symptom thereof, andthe like.

The phrases “systemic administration,” “administered systemically”,“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound of the invention(s), drugor other material, such that it enters the patient's system and, thus,is subject to metabolism and other like processes, for example,subcutaneous administration.

The language “therapeutically effective amount” of a compound of theinvention refers to an amount of an agent which is effective, uponsingle or multiple dose administration to the patient, treating orpreventing a neuropsychiatric disorder and/or symptoms of aneuropsychiatric disorder, or in prolonging the survivability of thepatient with such a neuropsychiatric disorder beyond that expected inthe absence of such treatment.

With respect to the nomenclature of a chiral center, terms “d” and “l”configuration are as defined by the IUPAC Recommendations. As to the useof the terms, diastereomer, racemate, epimer and enantiomer will be usedin their normal context to describe the stereochemistry of preparations.

2. Compounds Of The Invention

In one aspect, the invention provides compounds capable of modulating(e.g., inhibiting or stimulating) (directly or indirectly) 5-HT bindingactivity.

In one embodiment, the invention provides a compound capable ofagonizing 5-HT2c; and pharmaceutically acceptable esters, salts, andprodrugs thereof.

Certain preferred compounds include compounds specifically delineatedherein:

TABLE 1 Compounds Formula 1

APT-type PAT # R₁ R₂ 32 (5APT) C₆H₅ H 33 (CF₃-5APT) m-CF₃-C₆H₄ H 34(OCH₃-5APT) m-OCH₃-C₆H₄ 35 (Cl-5APT) m-Cl-C₆H₄ H 36 (6APT) H C₆H₅ 37(CF₃-6APT) H m-CF₃-C₆H₄ 38 (OCH₃-6APT) H m-OCH₃-C₆H₄ 39 (Cl-APT) Hm-Cl-C₆H₄ 40 (Br-6APT) H m-Br-C₆H₄

The invention also relates to the pharmaceutically acceptable salts andesters of the above-mentioned compounds.

Naturally occurring or synthetic isomers can be separated in severalways known in the art. Methods for separating a racemic mixture of twoenantiomers include chromatography using a chiral stationary phase (see,e.g., “Chiral Liquid Chromatography,” W. J. Lough, Ed. Chapman and Hall,New York (1989)). Enantiomers can also be separated by classicalresolution techniques. For example, formation of diastereomeric saltsand fractional crystallization can be used to separate enantiomers. Forthe separation of enantiomers of carboxylic acids, the diastereomericsalts can be formed by addition of enantiomerically pure chiral basessuch as brucine, quinine, ephedrine, strychnine, and the like.Alternatively, diastereomeric esters can be formed with enantiomericallypure chiral alcohols such as menthol, followed by separation of thediastereomeric esters and hydrolysis to yield the free, enantiomericallyenriched carboxylic acid. For separation of the optical isomers of aminocompounds, addition of chiral carboxylic or sulfonic acids, such ascamphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid canresult in formation of the diastereomeric salts.

According to another embodiment, the invention provides compounds whichassociate with or bind to GPCR or binding pocket thereof produced oridentified by the methods described herein.

3. Uses of the Compounds of the Invention

In one embodiment, the invention provides methods for treating a subjectfor a GPCR-mediated disorder, by administering to the subject aneffective amount of a compound capable of modulating (agonizing,antagonizing) a GPCR target. A GPCR disorder includes diseases anddisorders mediated by such GPCRs. The herein delineated compounds,compositions and methods are useful for treating or preventing disordersincluding, for example, neuropsychiatric disorders (e.g., obesity,addiction, anxiety, depression, schizophrenia, and sleep disorders),neurodegenerative disorders (e.g., Parkinson's Disease, Alzheimer'sDisease), neurological disorders (e.g., epilepsy), cardiovasculardisorders (e.g., 5-HT2b mediated disease, hypertension),gastrointestinal disorders (e.g., irritable bowel syndrome), andgenito-urinary tract disorders (e.g., bladder control). In certainembodiments, the subject is a mammal, e.g., a primate, e.g., a human.

In one embodiment, the invention provides compounds and methods fortreating a subject for a histamine (e.g., H1, H2, H3, H4)-mediateddisorder, by administering to the subject an effective amount of acompound capable of modulating (agonizing, antagonizing) a histaminetarget. A histamine disorder includes diseases and disorders mediated bysuch histamine (e.g., H1). The herein delineated compounds, compositionsand methods are useful for treating or preventing disorders including,for example, respiratory distress (e.g., bronchial constriction),diarrhea (GI contractions), edema, and hypotension (e.g., increasedvascular permeability), allergic response, and neuropsychiatric,neurodegenerative and neurological disorders herein.

In this embodiment, the compounds of the invention may either directlyor indirectly modulate (e.g., agonize, stimulate) the activity of 5-HT2cor specific domains thereof. A cell can be contacted with a compound ofthe invention to agonize 5-HT2c and modulate 5-HT2c mediated activity.Contacting cells or administering the compounds of the invention to asubject is one method of treating a cell or a subject suffering from orsusceptible to unwanted or undesired 5-HT2C mediated activity or a5-HT2c mediated disorder.

In one embodiment, a method of treating a subject suffering from orsusceptible to a 5-HT2c disorder includes administering to a subject inneed thereof a therapeutically effective amount of a compound capable ofdirectly or indirectly modulating the activity of 5-HT2c, to therebytreat the subject. Exemplary compounds include those compounds describedherein (e.g., APT, etc.).

Thus, in one embodiment, the invention provides methods for treating asubject for a 5-HT2C disorder, by administering to the subject aneffective amount of a compound capable of agonizing 5-HT2c.

In certain embodiments, the methods of the invention includeadministering to a subject a therapeutically effective amount of acompound of the invention in combination with another pharmaceuticallyactive compound. Other pharmaceutically active compounds that may beused can be found in Harrison's Principles of Internal Medicine,Thirteenth Edition, Eds. T. R. Harrison et al. McGraw-Hill N.Y., NY; andthe Physicians Desk Reference 50th Edition 1997, Oradell New Jersey,

Medical Economics Co., the complete contents of which are expresslyincorporated herein by reference. The compound of the invention and thepharmaceutically active compound may be administered to the subject inthe same pharmaceutical composition or in different pharmaceuticalcompositions (at the same time or at different times).

In certain embodiments, the compound of the invention can be used incombination therapy with conventional anti-obesity (e.g., fat absorptionblockers). Certain 5-HT drugs (e.g., 5-HTa or 5-HTb antagonists) have anundesirable side effect profile that tend to make them less then optimalor unsuitable for certain patients, that is, they demonstratecardiovascular (e.g., valvular heart disease, pulmonary hypertension,cardiotoxicity) or psychiatric undesirable and or life threatening sideeffect profiles.

Determination of a therapeutically effective amount or aprophylactically effective amount of the compound of the invention ofthe invention, can be readily made by the physician or veterinarian (the“attending clinician”), as one skilled in the art, by the use of knowntechniques and by observing results obtained under analogouscircumstances. The dosages may be varied depending upon the requirementsof the patient in the judgment of the attending clinician; the severityof the condition being treated and the particular compound beingemployed. In determining the therapeutically effective amount or dose,and the prophylactically effective amount or dose, a number of factorsare considered by the attending clinician, including, but not limitedto: the specific 5-HT2c disorder involved; pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration; the desired time course of treatment; the species ofmammal; its size, age, and general health; the specific diseaseinvolved; the degree of or involvement or the severity of the disease;the response of the individual patient; the particular compoundadministered; the mode of administration; the bioavailabilitycharacteristics of the preparation administered; the dose regimenselected; the kind of concurrent treatment (i.e., the interaction of thecompound of the invention with other co-administered therapeutics); andother relevant circumstances.

Treatment can be initiated with smaller dosages, which are less than theoptimum dose of the compound. Thereafter, the dosage may be increased bysmall increments until the optimum effect under the circumstances isreached. For convenience, the total daily dosage may be divided andadministered in portions during the day if desired. A therapeuticallyeffective amount and a prophylactically effective amount of a compoundof the invention of the invention is expected to vary from about 0.1milligram per kilogram of body weight per day (mg/kg/day) to about 100mg/kg/day.

Compounds determined to be effective for the prevention or treatment of5-HT2c disorders in animals, e.g., dogs, chickens, and rodents, may alsobe useful in treatment of 5-HT2c disorders in humans. Those skilled inthe art of treating 5-HT2c disease in humans will know, based upon thedata obtained in animal studies, the dosage and route of administrationof the compound to humans. In general, the dosage and route ofadministration in humans is expected to be similar to that in animals.

The identification of those patients who are in need of prophylactictreatment for 5-HT2C disorders is well within the ability and knowledgeof one skilled in the art. Certain of the methods for identification ofpatients which are at risk of developing 5-HT2C disorders which can betreated by the subject method are appreciated in the medical arts, suchas family history, and the presence of risk factors associated with thedevelopment of that disease state in the subject patient. A clinicianskilled in the art can readily identify such candidate patients, by theuse of, for example, clinical tests, physical examination andmedical/family history.

A method of assessing the efficacy of a treatment in a subject includesdetermining the pre-treatment extent of a 5-HT2C disorder by methodswell known in the art (e.g., determining level of markers for the 5-HT2Cdisorder) and then administering a therapeutically effective amount of acompound delineated herein according to the invention to the subject.After an appropriate period of time after the administration of thecompound (e.g., 1 day, 1 week, 2 weeks, one month, six months), theextent of the 5-HT2C disorder is determined again. The modulation (e.g.,decrease) of the extent or invasiveness of the 5-HT2C disorder indicatesefficacy of the treatment. The extent or invasiveness of the 5-HT2Cdisorder may be determined periodically throughout treatment. Forexample, the extent or invasiveness of the 5-HT2C disorder may bechecked every few hours, days or weeks to assess the further efficacy ofthe treatment. A decrease in extent or invasiveness of the 5-HT2Cdisorder indicates that the treatment is efficacious. The methoddescribed may be used to screen or select patients that may benefit fromtreatment with a modulating compound of a 5-HT2C disorder.

As used herein, “obtaining a biological sample from a subject,” includesobtaining a sample for use in the methods described herein. A biologicalsample is described above.

Yet another aspect presents a method to identify a compound thatmodulates the interaction of 5-HT2C or specific domains thereof. Themethod may include obtaining the crystal structure of 5-HT2C or specificdomains thereof (optionally apo form or complexed) or obtaining theinformation relating to the crystal structure of 5-HT2C or specificdomains thereof (optionally apo form or complexed), in the presenceand/or absence of the test compound. Compounds may then be computermodeled into or on the 5-HT2C structure, or specific domains thereof(e.g., a binding site of the crystal structure) to predict stabilizationof the interaction between the 5-HT2C or specific domains thereof andthe test compound. Once potential modulating compounds are identified,the compounds may be screened using cellular assays, such as the onesidentified herein and competition assays known in the art. Compoundsidentified in this manner are useful as therapeutic agents.

In another aspect, a compound of the invention is packaged in atherapeutically effective amount with a pharmaceutically acceptablecarrier or diluent. The composition may be formulated for treating asubject suffering from or susceptible to a 5-HT2C disorder, and packagedwith instructions to treat a subject suffering from or susceptible to a5-HT2C disorder.

In another aspect, the invention provides methods for modulating 5-HT2Cdisease. In one embodiment, a method of modulating 5-HT2C (or a 5-HT2Cdisorder) according to the invention includes contacting cells with acompound capable of modulating 5-HT2C (or a 5-HT2C disorder), orspecific domains thereof. In either embodiment, the contacting may be invitro, e.g., by addition of the compound to a fluid surrounding thecells, for example, to the growth media in which the cells are living orexisting. The contacting may also be by directly contacting the compoundto the cells. Alternately, the contacting may be in vivo, e.g., bypassage of the compound through a subject; for example, afteradministration, depending on the route of administration, the compoundmay travel through the digestive tract or the blood stream or may beapplied or administered directly to cells in need of treatment.

In another aspect, methods of inhibiting a 5-HT2C disorder in a subjectinclude administering an effective amount of a compound of the invention(i.e., a compound described herein) to the subject. The administrationmay be by any route of administering known in the pharmaceutical arts.The subject may have a 5-HT2C disorder, may be at risk of developing a5-HT2C disorder, or may need prophylactic treatment prior to anticipatedor unanticipated exposure to conditions capable of increasingsusceptibility to a 5-HT2C disorder.

In one aspect, a method of monitoring the progress of a subject beingtreated with a compound herein includes determining the pre-treatmentstatus (e.g., progression, target profile, Marker profile) of the 5-HT2Cdisorder, administering a therapeutically effective amount of a compoundherein to the subject, and determining the status (e.g., progression,target profile, Marker profile) of the 5-HT2C disorder after an initialperiod of treatment with the compound, wherein the modulation of thestatus indicates efficacy of the treatment.

The subject may be at risk of a 5-HT2C disorder, may be exhibitingsymptoms of a 5-HT2C disorder, may be susceptible to a 5-HT2C disorderand/or may have been diagnosed with a 5-HT2C disorder.

If the modulation of the status indicates that the subject may have afavorable clinical response to the treatment, the subject may be treatedwith the compound. For example, the subject can be administered atherapeutically effective dose or doses of the compound.

In another aspect, methods for evaluating a test compound comprisecontacting a 5-HT2C or specific domains thereof with a test compound(complex), and evaluating the binding interaction following contact,wherein a change in the stability of the complex relative to a referencevalue is an indication that the test compound modulates the stability ofthe complex.

The 5-HT2C or specific domains thereof complex may be modeled in silico,or may be a complex within a cell, isolated from a cell, recombinantlyexpressed, purified or isolated from a cell or recombinant expressionsystem or partially purified or isolated from a cell or recombinantexpression system.

Kits of the invention include kits for treating a 5-HT2C disorder in asubject. The kit may include a compound of the invention, for example, acompound described herein, pharmaceutically acceptable esters, salts,and prodrugs thereof, and instructions for use. The instructions for usemay include information on dosage, method of delivery, storage of thekit, etc. The kits may also include, reagents, for example, testcompounds, buffers, media (e.g., cell growth media), cells, etc. Testcompounds may include known compounds or newly discovered compounds, forexample, combinatorial libraries of compounds. One or more of the kitsof the invention may be packaged together, for example, a kit forassessing the efficacy of an treatment for a 5-HT2C disorder may bepackaged with a kit for monitoring the progress of a subject beingtreated for a 5-HT2C disorder according to the invention.

The present methods can be performed on cells in culture, e.g. in vitroor ex vivo, or on cells present in an animal subject, e.g., in vivo.Compounds of the inventions can be initially tested in vitro usingprimary cultures of cells, e.g., transformed cells, and the like.

The present method can be performed on cells in culture, e.g. in vitroor ex vivo, or on cells present in an animal subject, e.g., in vivo.Compounds of the invention can be initially tested in vitro using cellsfrom the respiratory tract from embryonic rodent pups (See e.g. U.S.Pat. No. 5,179,109—fetal rat tissue culture), or other mammalian (Seee.g. U.S. Pat. No. 5,089,517—fetal mouse tissue culture) ornon-mammalian animal models.

Alternatively, the effects of compounds of the invention can becharacterized in vivo using animals models.

4. Pharmaceutical Compositions

The invention also provides a pharmaceutical composition, comprising aneffective amount of a compound and a pharmaceutically acceptablecarrier. In a further embodiment, the effective amount is effective totreat a 5-HT2C disorder, as described previously.

In an embodiment, the compound of the invention is administered to thesubject using a pharmaceutically-acceptable formulation, e.g., apharmaceutically-acceptable formulation that provides sustained deliveryof the compound of the invention to a subject for at least 12 hours, 24hours, 36 hours, 48 hours, one week, two weeks, three weeks, or fourweeks after the pharmaceutically-acceptable formulation is administeredto the subject.

In certain embodiments, these pharmaceutical compositions are suitablefor topical or oral administration to a subject. In other embodiments,as described in detail below, the pharmaceutical compositions of thepresent invention may be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastes;(2) parenteral administration, for example, by subcutaneous,intramuscular or intravenous injection as, for example, a sterilesolution or suspension; (3) topical application, for example, as acream, ointment or spray applied to the skin; (4) intravaginally orintrarectally, for example, as a pessary, cream or foam; or (5) aerosol,for example, as an aqueous aerosol, liposomal preparation or solidparticles containing the compound.

The phrase “pharmaceutically acceptable” refers to those compound of theinventions of the present invention, compositions containing suchcompounds, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” includespharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier is “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions containing a compound of the invention(s) include thosesuitable for oral, nasal, topical (including buccal and sublingual),rectal, vaginal, aerosol and/or parenteral administration. Thecompositions may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. The amountof active ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the host beingtreated, the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect. Generally, out of one hundred per cent,this amount will range from about 1 per cent to about ninety-ninepercent of active ingredient, preferably from about 5 per cent to about70 per cent, more preferably from about 10 per cent to about 30 percent.

Methods of preparing these compositions include the step of bringinginto association a compound of the invention(s) with the carrier and,optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation a compound of the invention with liquid carriers, or finelydivided solid carriers, or both, and then, if necessary, shaping theproduct.

Compositions of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of theinvention(s) as an active ingredient. A compound may also beadministered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compound of theinvention(s) include pharmaceutically-acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

In addition to inert diluents, the oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compound of the invention(s) maycontain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Pharmaceutical compositions of the invention for rectal or vaginaladministration may be presented as a suppository, which may be preparedby mixing one or more compound of the invention(s) with one or moresuitable nonirritating excipients or carriers comprising, for example,cocoa butter, polyethylene glycol, a suppository wax or a salicylate,and which is solid at room temperature, but liquid at body temperatureand, therefore, will melt in the rectum or vaginal cavity and releasethe active agent.

Compositions of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof the invention(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound ofthe invention(s) may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition tocompound of the invention(s) of the present invention, excipients, suchas animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of theinvention(s), excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

The compound of the invention(s) can be alternatively administered byaerosol. This is accomplished by preparing an aqueous aerosol, liposomalpreparation or solid particles containing the compound. A nonaqueous(e.g., fluorocarbon propellant) suspension could be used. Sonicnebulizers are preferred because they minimize exposing the agent toshear, which can result in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically-acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the invention(s) to the body. Such dosageforms can be made by dissolving or dispersing the agent in the propermedium. Absorption enhancers can also be used to increase the flux ofthe active ingredient across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe active ingredient in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of the invention.

Pharmaceutical compositions of the invention suitable for parenteraladministration comprise one or more compound of the invention(s) incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers, which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofcompound of the invention(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compound of the invention(s) are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically-acceptable carrier.

Regardless of the route of administration selected, the compound of theinvention(s), which may be used in a suitable hydrated form, and/or thepharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels and time course of administration of the activeingredients in the pharmaceutical compositions of the invention may bevaried so as to obtain an amount of the active ingredient which iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic tothe patient. An exemplary dose range is from 0.1 to 10 mg per day.

A preferred dose of the compound of the invention for the presentinvention is the maximum that a patient can tolerate and not developserious side effects. Preferably, the compound of the invention of thepresent invention is administered at a concentration of about 0.001 mgto about 100 mg per kilogram of body weight, about 0.001-about 10 mg/kgor about 0.001mg-about 100 mg/kg of body weight. Ranges intermediate tothe above-recited values are also intended to be part of the invention.

6. Screening Methods and Systems

In another aspect, the invention provides a machine readable storagemedium which comprises the structural coordinates of either one or bothof the binding pockets identified herein, or similarly shaped,homologous binding pockets. Such storage medium encoded with these dataare capable of displaying a three-dimensional graphical representationof a molecule or molecular complex which comprises such binding pocketson a computer screen or similar viewing device.

-   -   The invention also provides methods for designing, evaluating        and identifying compounds which bind to the aforementioned        binding pockets. Thus, the computer produces a three-dimensional        graphical structure of a molecule or a molecular complex which        comprises a binding pocket.

In another embodiment, the invention provides a computer for producing athree-dimensional representation of a molecule or molecular complexdefined by structure coordinates of 5-HT2C or domains thereof, or athree-dimensional representation of a homologue of said molecule ormolecular complex, wherein said homologue comprises a binding pocketthat has a root mean square deviation from the backbone atoms of saidamino acids of not more than 2.0 (more preferably not more than 1.5)angstroms

In exemplary embodiments, the computer or computer system can includecomponents which are conventional in the art, e.g., as disclosed in U.S.Pat. Nos. 5,978,740 and/or 6,183,121 (incorporated herein by reference).For example, a computer system can includes a computer comprising acentral processing unit (“CPU”), a working memory (which may be, e.g.,RAM (random-access memory) or “core” memory), a mass storage memory(such as one or more disk drives or CD-ROM drives), one or morecathode-ray tube (CRT) or liquid crystal display (LCD) displayterminals, one or more keyboards, one or more input lines, and one ormore output lines, all of which are interconnected by a conventionalsystem bus.

Machine-readable data of this invention may be inputted to the computervia the use of a modem or modems connected by a data line. Alternativelyor additionally, the input hardware may include CD-ROM drives, diskdrives or flash memory. In conjunction with a display terminal, akeyboard may also be used as an input device.

Output hardware coupled to the computer by output lines may similarly beimplemented by conventional devices. By way of example, output hardwaremay include a CRT or LCD display terminal for displaying a graphicalrepresentation of a binding pocket of this invention using a programsuch as QUANTA or PYMOL. Output hardware might also include a printer,or a disk drive to store system output for later use.

In operation, the CPU coordinates the use of the various input andoutput devices, coordinates data accesses from the mass storage andaccesses to and from working memory, and determines the sequence of dataprocessing steps. A number of programs may be used to process themachine-readable data of this invention, includingcommercially-available software.

A magnetic storage medium for storing machine-readable data according tothe invention can be conventional. A magnetic data storage medium can beencoded with a machine-readable data that can be carried out by a systemsuch as the computer system described above. The medium can be aconventional floppy diskette or hard disk, having a suitable substratewhich may be conventional, and a suitable coating , which may also beconventional, on one or both sides, containing magnetic domains whosepolarity or orientation can be altered magnetically. The medium may alsohave an opening (not shown) for receiving the spindle of a disk drive orother data storage device.

The magnetic domains of the medium are polarized or oriented so as toencode in manner which may be conventional, machine readable data suchas that described herein, for execution by a system such as the computersystem described herein.

An optically-readable data storage medium also can be encoded withmachine-readable data, or a set of instructions, which can be carriedout by a computer system. The medium can be a conventional compact diskread only memory (CD-ROM) or a rewritable medium such as amagneto-optical disk which is optically readable and magneto-opticallywritable.

In the case of CD-ROM, as is well known, a disk coating is reflectiveand is impressed with a plurality of pits to encode the machine-readabledata. The arrangement of pits is read by reflecting laser light off thesurface of the coating. A protective coating, which preferably issubstantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, adata-recording coating has no pits, but has a plurality of magneticdomains whose polarity or orientation can be changed magnetically whenheated above a certain temperature, as by a laser. The orientation ofthe domains can be read by measuring the polarization of laser lightreflected from the coating. The arrangement of the domains encodes thedata as described above.

Structure data, when used in conjunction with a computer programmed withsoftware to translate those coordinates into the 3-dimensional structureof a molecule or molecular complex comprising a binding pocket may beused for a variety of purposes, such as drug discovery.

For example, the structure encoded by the data may be computationallyevaluated for its ability to associate with chemical entities. Chemicalentities that associate with a binding pocket of a 5-HT2C or specificdomains thereof, and are potential drug candidates. Alternatively, thestructure encoded by the data may be displayed in a graphicalthree-dimensional representation on a computer screen. This allowsvisual inspection of the structure, as well as visual inspection of thestructure's association with chemical entities.

Thus, according to another embodiment, the invention relates to a methodfor evaluating the potential of a chemical entity to associate with a) amolecule or molecular complex comprising a binding pocket of 5-HT2C orspecific domains thereof, or b) a homologue of said molecule ormolecular complex, wherein said homologue comprises a binding pocketthat has a root mean square deviation from the backbone atoms of saidamino acids of not more than 2.0 (more preferably 1.5) angstroms.

This method comprises the steps of:

i) employing computational means to perform a fitting operation betweenthe chemical entity and a binding pocket of the molecule or molecularcomplex; and

ii) analyzing the results of the fitting operation to quantify theassociation between the chemical entity and the binding pocket. The term“chemical entity”, as used herein, refers to chemical compounds,complexes of at least two chemical compounds, and fragments of suchcompounds or complexes.

The design of compounds that bind to 5-HT2C or specific domains thereofbinding pockets according to this invention generally involvesconsideration of several factors. First, the entity must be capable ofphysically and structurally associating with parts or all of the 5-HT2Cor specific domains thereof-related binding pockets. Non-covalentmolecular interactions important in this association include hydrogenbonding, van der Waals interactions, hydrophobic interactions andelectrostatic interactions. Second, the entity must be able to assume aconformation that allows it to associate with the 5-HT2C, or specificdomains thereof, binding pocket(s) directly. Although certain portionsof the entity will not directly participate in these associations, thoseportions of the entity may still influence the overall conformation ofthe molecule. This, in turn, may have a significant impact on potency.Such conformational requirements include the overall three-dimensionalstructure and orientation of the chemical entity in relation to all or aportion of the binding pocket, or the spacing between functional groupsof an entity comprising several chemical entities that directly interactwith the binding pocket or homologues thereof.

The potential binding effect of a chemical entity on a 5-HT2C orspecific domains thereof may be analyzed prior to its actual synthesisand testing by the use of computer modeling techniques. If thetheoretical structure of the given entity suggests insufficientinteraction and association between it and the target binding pocket,testing of the entity is obviated. However, if computer modelingindicates a strong interaction, the molecule may then be synthesized andtested for its ability to bind to a binding pocket. This may beachieved, e.g., by testing the ability of the molecule modulate 5-HT2C,or specific domains thereof, binding activity, e.g., using assaysdescribed herein or known in the art. In this manner, synthesis ofinoperative compounds may be avoided.

A potential binder of 5-HT2C or specific domains thereof (e.g., bindingpocket) may be computationally evaluated by means of a series of stepsin which chemical entities or fragments are screened and selected fortheir ability to associate with the 5-HT2C or specific domainsthereof-related binding pockets.

One skilled in the art may use one of several methods to screen chemicalentities or fragments for their ability to associate with a 5-HT2C orspecific domains thereof -related binding pocket. This process may beginby visual inspection of, for example, 5-HT2C or specific domainsthereof-related binding pocket on the computer screen based on the5-HT2C, or specific domains thereof structure coordinates describedherein, or other coordinates which define a similar shape generated fromthe machine-readable storage medium. Selected fragments or chemicalentities may then be positioned in a variety of orientations, or docked,within that binding pocket as defined supra. Docking may be accomplishedusing software such as Quanta and DOCK, followed by energy minimizationand molecular dynamics with standard molecular mechanics force fields,such as CHARMM and AMBER.

Specialized computer programs (e.g., as known in the art and/orcommercially available and/or as described herein) may also assist inthe process of selecting fragments or chemical entities.

Once suitable interacting/binding compound chemical entities orfragments have been selected, they can be assembled into a singlecompound or complex. Assembly may be preceded by visual inspection ofthe relationship of the fragments to each other on the three-dimensionalimage displayed on a computer screen in relation to the structurecoordinates of the target binding pocket.

Instead of proceeding to build an of a binding pocket in a step-wisefashion one fragment or chemical entity at a time as described above,interacting or other binding compounds may be designed as a whole or “denovo” using either an empty binding site or optionally including someportion(s) of a known interacting/binding compound(s). There are many denovo ligand design methods known in the art, some of which arecommercially available (e.g., LeapFrog, available from TriposAssociates, St. Louis, Mo.).

Other molecular modeling techniques may also be employed in accordancewith this invention [see, e.g., N. C. Cohen et al., “Molecular ModelingSoftware and Methods for Medicinal Chemistry”, J. Med. Chem., 33, pp.883-894 (1990); see also, M. A. Navia and M. A. Murcko, “The Use ofStructural Information in Drug Design”, Current Opinions in StructuralBiology, 2, pp. 202-210 (1992); L. M. Balbes et al., “A Perspective ofModern Methods in Computer-Aided Drug Design”, in Reviews inComputational Chemistry, Vol. 5, K. B. Lipkowitz and D. B. Boyd, Eds.,VCH, New York, pp. 337-380 (1994); see also, W. C. Guida, “Software ForStructure-Based Drug Design”, Curr. Opin. Struct. Biology, 4, pp.777-781 (1994)].

Once a compound has been designed or selected, the efficiency with whichthat entity may bind to a binding pocket may be tested and optimized bycomputational evaluation.

Specific computer software is available in the art to evaluate compounddeformation energy and electrostatic interactions. Examples of programsdesigned for such uses include: AMBER; QUANTA/CHARMM (Accelrys, Inc.,Madison, Wis.) and the like. These programs may be implemented, forinstance, using a commercially-available graphics workstation. Otherhardware systems and software packages will be known to those skilled inthe art.

Another technique involves the in silico screening of virtual librariesof compounds, e.g., as described herein. Many thousands of compounds canbe rapidly screened and the best virtual compounds can be selected forfurther screening (e.g., by synthesis and in vitro testing). Smallmolecule databases can be screened for chemical entities or compoundsthat can bind, in whole or in part, to a GPCR, 5-HT2c, or specificdomains thereof. In this screening, the quality of fit of such entitiesto the binding site may be judged either by shape complementarity or byestimated interaction energy.

The compounds herein are also advantageous in that they possess a higherlevel of structural rigidity relative to previously reported 5-HT2active compounds. This advantage is useful for probing structuralinformation about GPCR targets (e.g., histamine, serotonin, muscarinic,etc.) and sub-families thereof. Such information is useful inelucidating binding pocket information, homology, sequence, etc. Thus,in aspects, the invention includes the use of the compounds delineatedherein as probes for elucidation of target structure information. Theuse includes methods for studying interaction and function of thecompounds herein with a PGCR target (both using “wet lab” experimentalassays, probes, crystallization techniques and protocols, proteinstudies, as well as in silico methods using representations of thecompounds).

EXAMPLES

The invention is further illustrated by the following examples which areintended to illustrate but not limit the scope of the invention.

Example 1 Database of Small Molecules

The NCI/DTP maintains a repository of approximately 240,000 samples(i.e., the plated compound set) which are non-proprietary and offered tothe research community for discovery and development of new agents forthe treatment of cancer, AIDS, or opportunistic infections afflictingsubjects with cancer or AIDS. The three-dimensional coordinates for theNCI/DTP plated compound set is obtained in the MDL SD format(chm.tu-dresden.de/edv/vamp65/REFERS/vr_03d.htm) and converted to themo12 format by the DOCK utility program SDF2MOL2. Partial atomiccharges, solvation energies and van der Waals parameters for the ligandsare calculated using SYBDB and added to the plated compound set mol2files.

Example 2 Database Screening To Identify Potential Small MoleculeInteracting/Binding Compounds.

In lieu of conducting high-throughput screening, a more rapid andeconomical structure-based approach combining molecular docking insilico with functional testing is used. A large chemical library ofcompounds with known three-dimensional structure is positioned in thestructural pocket selected by SPHGEN (UCSF) on the crystal structure ofa GPCR. This approach combines resources available through the NCI/DTP(atomic coordinates and small molecules) with improved molecular dockingand scoring algorithms imposed in DOCK5.1 (UCSF). 20,000 small moleculecompounds with drug-like characteristics (following the Lipinski rules)were docked into the 5-HT2c crystal structure in 100 differentorientations using DOCK5.1. The compounds with the highest scores arerequested for functional testing from the NCI/DTP.

The National Cancer Institute/Developmental Therapeutics Program(NCI/DTP) maintains a repository of approximately 220,000 samples (theplated compound set) that are nonproprietary and offered to theextramural research community free of charge. The three-dimensionalcoordinates for the NCI/DTP plated compound set was obtained in the MDLSD format and converted to the mo12 format by the DOCK utility programSDF2MOL2. Partial atomic charges, solvation energies, and van der Waalsparameters for the ligands were calculated using SYBDB and added to theplated compound set mol2 file.In Silico Molecular Docking of Potential Small MoleculeInteracting/binding compounds. All docking calculations are performedwith the DOCK, v5.1.0. The general features of DOCK include rigidorienting of ligands to receptor spheres, AMBER energy scoring, GB/SAsolvation scoring, contact scoring, internal nonbonded energy scoring,ligand flexibility, and both rigid and torsional simplex minimization.Unlike previously distributed versions, this release incorporatesautomated matching, internal energy (used in flexible docking), scoringfunction hierarchy, and new minimizer termination criteria. Thecoordinates for the molecular model of the 5-HT2c domain are used in themolecular docking calculations. To prepare the site for docking, allwater molecules are removed. Protonation of receptor residues isperformed with Sybyl (Tripos, St. Louis, Mo.). The structure is exploredusing sets of spheres to describe potential binding pockets. The numberof orientations per molecule is 100. Intermolecular AMBER energy scoring(vdw+columbic), contact scoring, and bump filtering are implemented inDOCK5.1.0. SETOR and GRASP are used to generate molecular graphicimages.

As shown herein, representative compounds herein have GPCR modulatingactivity.

Example 3 Chemicals

[³H]-Ketanserin (specific activity 72.2 Ci/mmol) andmyo-[2-³H(N)]-Inositol (specific activity 18.5 Ci/mmol) were purchasedfrom Perkin-Elmer Life Science (Boston, Mass.) and[N⁶-methyl-³H]-mesulergine (specific activity 72.0 Ci/mmol) fromAmersham Biosciences (GE healthcare, Piscataway, N.J.). Other compoundswere obtained in highest purity from Sigma-Aldrich (St. Louis, Mo.).

Clonal Cell Culture and Transfection

All cell lines were maintained by following ATCC suggestion, ChineseHamster Ovary cells (CHO-K1, ATCC CCL-61) in Ham's F-12 mediumsupplemented with 10% fetal bovine serum, 1% sodium bicarbonate(Mediatech 25-035-CI), 10 IU/ml Penicillin and 10 ug/ml Streptomycin,and human embryonic kidney (HEK) 293 in minimum essential medium (Eagle)(MEM) with 2 mM L-glutamine adjusted to contain 1.5 g/L sodiumbicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodiumpyruvate (90%) with 10% fetal bovine serum, 10 IU/ml Penicillin and 10ug/ml Streptomycin. Cells were grown at 37° C. in a humidified incubatorwith 5% CO2. The cDNAs encoding the human 5-HT_(2A), 5-HT_(2B), and5HT_(2C) receptors (wild type) were purchased from UMR Rolla, Mo.) fortransient transfection of the clonal cells. For radioreceptor bindingassays, 5-HT_(2A), 5-HT_(2B), and 5-HT_(2C) receptor membranes wereprepared from transfected CHO-K1 cells. For functional assays measuringactivity of PLC/IP formation, transfected CHO-K1 cells were used for5-HT_(2A) and 5HT_(2C) receptors. For 5HT_(2B) receptors, however, morerobust and consistent results for the PLC/IP assay were obtained usingtransfected HEK cells (Setola et al., 2005). Twenty-four hours beforetransfection, cells were seeded at 40% confluence in 100 mm dishes forradioreceptor binding assays or at 10⁵ cells per well in 12-well platesfor functional assays. CHO-K1 cells were transiently transfected with 12μg of plasmid and 32 μl of lipofectamine (Invitrogen) per 100 mm dishfor radioreceptor binding assays, or, 0.8 μg plasmid and 4.0 μl oflipofectamine per well for functional assays. For 5-HT_(2B) functionalassays using HEK cells, 24 μg plasmid DNA was mixed with 60 μl of

Lipofectamine 2000 (Invitrogen) to transfect 1-2×10⁶ cells in a 10-cmplate. Cells were allowed to express transfected receptors for another24 hrs (Herrick, 1997).

Radioreceptor Assays

Radioreceptor saturation and competition binding assays were performedusing membrane homogenates, similar to our methods reported previouslyfor the phylogenetically closely related histamine H₁ GPCR (Booth, 2002;Moniri et al., 2004). [³H]-Ketanserin was used to radiolabel HT_(2A)receptors and [³H]-mesulergine for 5-HT_(2B) and 5HT_(2C) receptors.Briefly, forty-eight hours following CHO cell transfection, cells wereharvested and homogenized in 50 mM Tris-HCl containing 0.1% ascorbicacid and 4.0 mM CaCl₂ at pH 7.4 (assay buffer). The homogenate wascentrifuged at 35,000 g for 25 min and the resulting membrane pellet wasre-suspended in assay buffer. Protein concentration was determined bythe method of Lowry et al. (Lowry, 1951). For saturation binding assays,membrane suspension containing 100 μg protein was incubated with 0.1-5.0nM [³H]-ketanserin (5-HT_(2A) receptors) or 0.1-20 nM [³H]-mesulergine(5-HT_(2B) and 5HT_(2C) receptors) in a total assay buffer volume of 250μl. Non-specific binding was determined in the presence of 10 μMmethysergide (5-HT_(2A) receptors) or 1.0 μM mianserin (5-HT_(2B) and5HT_(2C) receptors). Competition binding assays were conducted similarlywith 1.0 nM [³H]-ketanserin or [³H]-mesulergine. Incubation ofradioreceptor binding assay mixtures was for 1.0 h at 37° C., withtermination by rapid filtration through Whatman GF/B filters using a96-well cell harvester (Tomtec, Hamden, Conn.). The membrane-bound[³H]-radioligand retained on the filter discs was quantified by liquidscintillation spectrometry. Data were analyzed by nonlinear regressionusing the sigmoidal curve-fitting algorithms in Prism 4.03 (GraphPadSoftware Inc., San Diego, Calif.). Ligand affinity is expressed as anapproximation of K₁ values by conversion of the IC₅₀ data to K_(0.5)values using the equation K_(0.5)=IC₅₀/1+L/K_(D), where L is theconcentration of radioligand having affinity K_(D) (Cheng, 1973). Eachexperimental condition was performed in triplicate and each experimentwas performed a minimum of three times to determine S.E.M.

Measurement of [³H]-IP Formation in CHO-K1 and HEK Cells

Functional activation of PLC was measured as [³H]-IP formation in CHOcells transiently expressing 5-HT_(2A) or 5HT_(2C) receptors and HEKcells transiently expressing 5HT_(2B) receptors, as previously reportedby our lab (Booth, 2002; Moniri et al., 2004). Briefly, thirty-two hoursfollowing transfection, cells in inositol-free Dulbecco's modifiedEagle's medium (DMEM) were labeled with 1 μCi/ml myo-[2-³H]-inositol, aprecursor of the PLC-β substrate phosphatidylinositol. Cells then werewashed and incubated in DMEM containing 25 mM Hepes (pH 7.4), 10 mMLiCl, 10 μM pargyline (with addition of 5% dialyzed FBS for HEK cells),and various concentrations of test ligand for 45-60 min at 37° C. Afteraspiration of media, wells were placed on ice and lysed by incubationwith 50 mM formic acid (15-60 min). Formic acid was neutralized withammonium hydroxide and all contents from each well were added toindividual AG1-X8 200-400 formate resin anion exchange columns. Ammoniumformate/formic acid (1.2 M/0.1 M) was used to elute [³H]-IP directlyinto scintillation vials for counting of tritium by liquid scintillationspectrometry. Resulting data were analyzed using the nonlinearregression algorithms in Prism 4.03 and are expressed as mean percentageof control [³H]-IP formation, with potency expressed as concentrationrequired to produce 50% maximal [³H]-IP formation (EC₅₀)±S.E.M (n≥3).

Example 4 Radioreceptor Assays

Radioligand saturation binding analysis of 5HT-subtype receptors: Therewas no measurable specific radioligand binding using membranes preparedfrom null-transfected CHO and HEK cells. Using membranes prepared fromCHO cells transiently transfected with 5-HT_(2A), 5-HT_(2B), or 5HT_(2C)cDNA, however, saturable specific radioligand bindingoccurs—representative binding curves for [³H]-ketanserin labeled5HT_(2A) receptors and [³H]-mesulergine labeled 5-HT_(2B) receptors and5HT_(2C) receptors are shown in FIGS. 2A-C. [³H]-Ketanserin binds to anapparent single population of 5HT_(2A) receptors (B_(max)=1.73±0.11pmol/mg protein) with high affinity (K_(D)=0.80±0.03 nM). Similarly,[³H]-mesulergine labels a single population of 5HT_(2B) receptors withB_(max)=1.13±0.39 pmol/mg protein and K_(D)=5.19±0.36 nM.[³H]-mesulergine also labels an apparent single population of 5HT_(2C)receptors (B_(max)=8.37±0.15 pmol/mg prot) with high affinity(K_(D)=0.88±0.03 nM).

Example 5

Competition binding analysis to determine (-)-trans-PAT 5HT₂-subtypeReceptor affinity. Representative 5-HT_(2A), 5-HT_(2B), and 5HT_(2C)radioligand displacement curves for (-)-trans-PAT are shown in FIG. 3.Curves are sigmoidal in shape and span 3-4 log ligand concentrationunits to achieve complete radioligand displacement, characteristic ofcompetitive displacement of ˜K_(D) radioligand concentration from asingle population of GPCRs. The K_(i)±SEM values for (-)-trans-PAT at5HT_(2A), 5-HT_(2B), and 5HT_(2C) receptors are 410±38, 130±28, and37.6±3.02 nM (respectively), with corresponding n_(H) values of 1.1±0.1,1.1±0.1, and 0.9±0.1.

Example 6 Functional Assays

Assessment of (-)-trans-PAT agonist activity at 5HT2-subtype receptors:The 5HT2 GPCR family is constitutively active when expressed in CHO andHEK cells, thus, functional activity here is reported relative to basalactivity of PLC/[³H]-IP formation in FIG. 4. In lysates ofnull-transfected CHO and HEK cells, no increase in basal activity ofPLC/[³H]-IP formation was detected after incubation with up to 10 μM5-HT for 45 min. In CHO cells transiently transfected with human5HT_(2C) cDNA, however, 5-HT produces a concentration-dependent increasein basal activity of PLC/[³H]-IP formation, with EC₅₀=6.30±0.55nM=1.3±0.2) and E_(max)˜0.1 μM (˜475% basal control activity), as shownin the FIG. 4 inset. Relative to the endogenous agonist, (-)-trans-PATis a full-efficacy 5HT_(2C) agonist that produces aconcentration-dependent increase in basal activity of PLC/[³H]-IPformation, with EC₅₀=21.4±2.22 nM=0.66±0.11) and E_(max)˜10 (˜475% basalcontrol activity) (FIG. 4). In CHO cells transiently transfected withhuman 5HT_(2A) cDNA, however, (-)-trans-PAT did not stimulatePLC/[³H]-IP formation at concentrations up to 10 μM; for comparison, 5HTEC₅₀=30±2 nM, E_(max)=1.0 μM (˜300% basal control activity) (data notshown). Likewise, in HEK cells transiently transfected with human5HT_(2B) cDNA, trans-PAT did not stimulate PLC/[³H]-IP formation atconcentrations up to 30 μM; for comparison, 5HT EC₅₀=19.7±9.21 nM,E_(max)˜1.0 μM (˜900% basal control activity (data not shown).

Example 7

Assessment of (-)-trans-PAT antagonist activity at 5HT2-subtypereceptors: Given that (-)-trans-PAT binds with moderate affinity to5HT_(2A) and 5HT_(2B) receptors (FIG. 3) but does not activate these5-HT2 receptor subtypes (FIG. 4), the ability of (-)-trans-PAT to act asa 5HT_(2A) and 5HT_(2B) receptor antagonist regarding 5-HT-mediatedstimulation of PLC/[³H]-IP formation was assessed and results are shownin FIGS. 5A-5B. In CHO cells expressing human 5HT_(2A) receptors, 5-HT(1.0 μM) stimulated PLC/[³H]-IP formation (˜250% basal control) and thiseffect was fully blocked by (-)-trans-PAT (10 μM) (FIG. 5A). In HEKcells expressing human 5HT_(2B) receptors, 5-HT (0.01 μM) stimulatedPLC/[³H]-IP formation (˜350% basal control) and this effect was fullyblocked by (-)-trans-PAT (3.0 μM) (FIG. 5B).

Example 8 Discussion

Results indicate that (-)-trans-PAT demonstrates full-efficacy foractivation of human 5HT_(2C) receptors as well as antagonist activity at5HT_(2A) and 5HT_(2B) receptors. These results have promisingimplications for development of novel 5HT_(2C)-based pharmacotherapy.For example, activation of brain 5HT_(2C) receptors is well establishedto be effective pharmacotherapy for obesity (Tecott et al., 1995;Vickers et al., 1999; 2001; Heisler et al., 2002). Meanwhile, therecurrently is no effective pharmacotherapy for cocaine addiction. It isthought, however, that disorders involving over-eating and drugself-administration are members of the same group of compulsivebehavioral disorders directed toward different objects, food and drugs(Simansky, 2005). Accordingly, a role for brain 5HT_(2C) receptoractivation in pharmacotherapy of both obesity and cocaine addictionappears logical. In fact, the balance of studies using reliable 5HT₂subtype-selective antagonists suggest that the reinforcing effects ofcocaine are reduced by 5HT_(2C) activation, and, discriminative stimulusand reinstating effects of cocaine are sensitive to attenuation by5HT_(2C) activation as well as by 5HT_(2A) antagonism (Bubar andCunningham, 2006).

In contrast, 5HT_(2A) receptor activation is closely associated withpsychomimetic activity (Nichols, 2004), as perhaps best demonstrated bythe inverse observation that drugs with 5HT_(2A) antagonist activity areeffective to treat psychosis and related neuropsychiatric disorders(Baldessarini and Tarazi, 2006). Thus, the possibility of 5HT_(2A)receptor activation concomitant with 5HT_(2C) activation for drugs thatshow only modest 15-fold selectivity in vitro, such as lorcaserin(Jensen, 2006; Smith et al., 2006), should be carefully considered inview of the sometimes subtle and complex nature of psychiatricdisturbances. Perhaps, the life-threatening cardiac valvulopathy andpulmonary hypertension associated with fenfluramine has led to a higherthreshold for selectivity (e.g., 100-fold, in vitro) regardingdevelopment of drugs that activate both 5HT_(2C) and 5HT_(2B) receptors.Nevertheless, clinical use of a diet drug by perhaps 30-50 millionpeople (Nilsson, 2006; Smith et al., 2006), often chronically, oftenunsupervised, could lead to problems if the drug demonstrates anyactivation of 5HT_(2A) and/or 5HT_(2B) receptors. We have made an extraeffort to ensure that any lead compounds we put forward for drugdevelopment demonstrate unequivocal 5HT_(2B) antagonism by switching toHEK cells because this clonal cell line gives more robust andreproducible functional results compared to CHO cells for the 5HT_(2B)receptor. Thus, a 5HT_(2C) agonist such as (-)-trans-PAT, thatdemonstrates unequivocal 5HT_(2A) and 5HT_(2B) antagonism, and is asmall lipophilic molecule that penetrates mammalian brain afterperipheral administration, appears to be well-suited for considerationas novel pharmacotherapy for obesity, cocaine addiction, psychosis,anxiety, and, perhaps other neuropsychiatric disorders.

Example 9

1g. Affinity of APT Analogs (Table 2) for 5HT_(2C) Receptors (FIG. 6).

Our laboratory also has completed 5HT_(2C) affinity experiments for APTs32 (SAPT), 36 (6APT), and 39 (C1-6APT) in Table 2. The radioligandcompetition displacement curves are shown in FIG. 6; the 5HT_(2C) Kivalues (±SEM) are 320±11, 1,800±23, and 316±15 nM, respectively.Although the n_(H) values are <1.0 (suggestive of agonist activity),5HT_(2C) affinity of these analogs is poor (Ki>300 nM).

These data suggest that moving the pendant phenyl ring from the1-position of the PAT molecular scaffold (Table 1) to the 5- or6-position of the APT scaffold (Table 2) diminishes affinity for5HT_(2C) receptors. Mutational analysis and molecular modeling studiesof 5HT2 and other aminergic neurotransmitter GPRCs suggest important π-πstacking binding interactions occur between the phenyl moieties ofligands and receptor amino acids in TMDs 5 and 6 (e.g., Roth et al.,1997; Wieland et al., 1999; Shapiro et al., 2000; Elz et al., 2000; Shiet al., 2002; Wetkaemper & Glennon, 2002). In addition to theseradioreceptor affinity results, preliminary functional and molecularmodeling results below suggest that PAT phenyl moiety orientation andsubstitution is key to providing potent and selective ligands thatactivate 5HT_(2C) receptors and not 5HT_(2A) and 5HT_(2B) receptors.

Example 10

2c. Functional Activity of APTs at 5HT2 Receptors (FIG. 7)

Preliminary data is available from the PDSP (2005) regarding functionalactivity of SAPT and C1-6APT (Table 2) at 5HT_(2C) vs. 5HT_(2A)receptors. SAPT is a moderate potency partial agonist at 5HT_(2C);EC₅₀˜80 nM; E_(max)˜75% 5HT value (5HT E_(max)˜80,000 cpm; not shown).Thus, the location of the pendant phenyl ring appears to have asignificant effect on ability of PAT analogs to activate 5HT_(2C)receptors—potency decreases as the phenyl moiety is moved from the1-position of (-)-trans-PAT (EC₅₀˜20 nM, FIG. 4), to the 5-position ofSAPT (EC₅₀˜80 nM, FIG. 7), to the 6-position of C1-6APT (EC₅₀˜4 μM). Atconcentrations to 10 μM, 5APT and C1-6APT did not activate 5HT_(2A)receptor-mediated PLC/[³H]-IP formation (PDSP, 2005; not shown).

Example 11

Affinity for 5-HT2c: Representative Compound Results

Ki @ APT-type 5HT_(2C) ± PAT # SEM; nM 32 (5APT)  320 ± 11 33 (CF₃- ~1905APT) 35 (Cl-5APT) ~220 36 (6APT) 1800 ± 23 37 (CF₃- ~1500 6APT) 39(Cl-6APT)  316 ± 15 40 (Br-6APT)  540 ± 10

Example 12

Synthesis of APT compounds has been previously reported in Booth et al.,Bioorg. Med. Chem., 14, (2006) 6640-6658, herein incorporated byreference in its entirety.

Functional Activity of APTs at 5HT₂ Receptors (FIG. 8)

Preliminary data is available from the PDSP (2005) regarding functionalactivity of 5APT and C1-6APT (Tables and structures herein) at 5HT_(2C)vs. 5HT_(2A) receptors. 5APT is a moderate potency partial agonist at5HT_(2C); EC₅₀˜80 nM; E_(max)˜75% 5HT value (5HT E_(max)˜80,000 cpm; notshown). Thus, the location of the pendant phenyl ring appears to have asignificant effect on ability of PAT analogs to activate 5HT_(2C)receptors—potency decreases as the phenyl moiety is moved from the1-position of (-)-trans-PAT (EC₅₀˜20 nM, FIG. 4), to the 5-position of5APT (EC₅₀˜80 nM, FIG. 8), to the 6-position of C1-6APT (EC₅₀˜4 μM). Atconcentrations to 10 μM, 5APT and C1-6APT did not activate 5HT_(2A)receptor-mediated PLC/[³H]-IP formation (PDSP, 2005; not shown).

REFERENCES

Arjona A A, Pooler A M, Lee R K, Wurtman R J. Effect of a 5HT_(2C)serotonin agonist, dexnorfenfluramine, on amyloid precursor proteinmetabolism in guinea pigs. Brain Res. 2002 951:135-140.Baldessarini R J, Tarazi F I. Pharmacotherapy of Psychosis and mania.In: Brunton L L, Laxo J S, Parker K L, eds. The Pharmacological Basis ofTherapeutics. 11th ed. New York: McGraw-Hill, 2006:461-500.Bubar M J, Cunningham K A. Distribution of serotonin 5-HT(2C) receptorsin the ventral tegmental area. Neuroscience. 2007 (doi:10.1016/j.neuroscience.2006.12.071).Bubar M J, Cunningham K A. Serotonin 5-HT_(2A) and 5-HT2C receptors aspotential targets for modulation of psychostimulant use and dependence.Current Topics and Medicinal Chemistry 2006; 6:1971-1985.Connolly H M, Crary J L, McGoon M D, Hensrud D D, Edwards B S, Edwards WD, Schaff H V. Valvular heart disease associated withfenfluramine-phentermine. N Engl J Med. 1997;337:581-5888. Erratum in: NEngl J Med 1997;337:1783.Fitzgerald L W, Burn T C, Brown B S, Patterson J P, Corjay M H,Valentine P A, Sun J H, Link J R, Abbaszade I, Hollis J M, et al.Possible role of valvular serotonin 5-HT(2B) receptors in thecardiopathy associated with fenfluramine. Mol Pharmacol 2000 57: 75-81.Fletcher P J, Grottick A J, Higgins G A. Differential effects of the5-HT(2A) receptor antagonist M100907 and the 5-HT(2C) receptorantagonist SB242084 on cocaine-induced locomotor activity, cocaineself-administration and cocaine-induced reinstatement of responding.Neuropsychopharmacology 2002 27:576-586.Frank M G, Stryker M P, Tecott L H. Sleep and sleep homeostasis in micelacking the 5-HT2c receptor. Neuropsychopharmacology. 2002 27:869-873.Giorgetti M, Tecott L H. Contributions of 5-HT(2C) receptors to multipleactions of central serotonin systems. Eur J Pharmacol. 2004 488:1-9.Heisler L K, Chu H M, Tecott L H. Epilepsy and obesity in serotonin5-HT2C receptor mutant mice. Ann N Y Acad Sci. 1998 861:74-78.Heisler L K, Cowley M A, Tecott L H, Fan W, Low M J, Smart J L,Rubinstein M, Tatro J B, Marcus J N, Holstege H, et al. Activation ofcentral melanocortin pathways by fenfluramine. Science (Wash, D.C.) 2002297: 609-611.Heisler L K, Tecott L H. A paradoxical locomotor response in serotonin5-HT(2C) receptor mutant mice. J Neurosci. 2000 20:RC71.Heisler L K, Zhou L, Bajwa P, Hsu J, Tecott L H Serotonin 5-HT(2C)receptors regulate anxiety-like behavior. Genes Brain Behav. 2007 (DOI10.1111/j.1601-183X.2007.00316.x)Jensen M D. Potential role of new therapies in modifying cardiovascularrisk in overweight patients with metabolic risk factors. Obesity. 200614:143S-149S.Julius D, Huang K N, Livelli T J, Axel R, Jessel T M. The 5HT2 receptordefines a family of structurally distinct but functionally conservedserotonin receptors. Proc. Natl. Acad. Sci. 1990 87:928-932.Julius D, MacDermott A B, Axel R, Jessell T M. MolecularCharacterization of a functional cDNA encoding the serotonin 1creceptor. Science 1988 241:558-564.Kennett G A, Pittaway K, Blackburn T P: Evidence that 5-HT2C receptorantagonists are anxiolytic in the rat Geller-Seifter model of anxiety.Psychopharmacology (Berl.) (1994) 114:90-96.Launay J M, Herve P, Peoc'h K, Tournois C, Callebert J, Nebigil C G,Etienne N, Drouet L, Humbert M, Simonneau G, et al. Function of theserotonin 5-hydroxytryptamine 2B receptor in pulmonary hypertension. NatMed 2002 8: 1129-1135.Marquis K L, Sabb A L, Logue S F, Brennan J A, Piesla M J, Comery T A,Grauer S M, Ashby C R Jr, Nguyen H Q, Dawson L A, Barrett J E, Stack G,Meltzer H Y, Harrison B L, Rosenzweig-Lipson S. WAY-163909[(7bR,10aR)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole]:A novel 5-hydroxytryptamine 2C receptor-selective agonist withpreclinical antipsychotic-like activity. J Pharmacol Exp Ther. 2007320:486-496.Muller C P, Huston J P. Determining the region-specific contributions of5-HT receptors to the psychostimulant effects of cocaine. TrendsPharmacol Sci. 2006 27:105-112.Nichols D E. Hallucinogens, Pharmacol. Ther. 2004 101:131-181.Nilsson B M. 5-Hydroxytryptamine 2C (5-HT2C) receptor agonists aspotential antiobesity agents. J Med Chem. 2006 49:4023-4034.Palvimaki E P, Roth B L, Majasuo H, Laakso A, Kuoppamaki M, Syvalahti E,Hietala J. Interactions of selective serotonin reuptake inhibitors withthe serotonin 5-HT2c receptor. sychopharmacology (Berl). 1996126:234-240.Pouwels H M, Smeets J L, Cheriex E C, Wouters E F. Pulmonaryhypertension and fenfluramine. Eur Respir J. 1990 May;3(5):606-7.Raymond J R. Mukhin Y V. Gelasco A. Turner J. Collinsworth G. Gettys TW. Grewal J S. Garnovskaya M N. Multiplicity of mechanisms of serotoninreceptor signal transduction. Pharmacol Ther. 2001 92:179-212.Reynolds G P, Yao Z, Zhang X, Sun J, Zhang Z. Pharmacogenetics oftreatment in first-episode schizophrenia: D3 and 5-HT2C receptorpolymorphisms separately associate with positive and negative symptomresponse. Eur Neuropsychopharmacol. 2005 March;15(2):143-51.Rocha B A, Goulding E H, O'Dell L E, Mead A N, Coufal N G, Parsons L H,Tecott L H. Enhanced locomotor, reinforcing, and neurochemical effectsof cocaine in serotonin 5-hydroxytryptamine 2C receptor mutant mice. JNeurosci. 2002 ;22:10039-10045.Rosenzweig-Lipson S, Sabb A, Stack G, Mitchell P, Lucki I, Malberg J E,Grauer S, Brennan J, Cryan J F, Sukoff Rizzo S J, Dunlop J, Barrett J E,Marquis K L. Antidepressant-like effects of the novel, selective,5-HT(2C) receptor agonist WAY-163909 in rodents. Psychopharmacology(Berl). 2007 192:159-170.Roth B L. Drugs and valvular heart disease. N Engl J Med. 2007; 356:6-9.Rothman R B, Baumann M H, Savage J E, Rauser L, McBride A, Hufeisen S J,and Roth B L. Evidence for possible involvement of 5-HT(2B) receptors inthe cardiac valvulopathy associated with fenfluramine and otherserotonergic medications. Circulation 2000 102: 2836-2841.Saltzman A G, Morse B, Whitman M M, Ivanshchenko Y, Jaye M, Felder S.Cloning of the human serotonin 5-HT2 and 5-HT1C receptor subtypes.Biochem Biophys Res Commun. 1991 181:1469-7148.Sanders-Bush E, Mayer S E. Serotonin Receptor Agonists and Antagonists.Chapter 11, in Goodman and Gilman's The Pharmacological Basis ofTherapeutics 11^(th) Edition. Brunton L L, Lazo J S, Parker K L,Editors, McGraw-Hill, New York, 297-315, 2006.Sard H, Kumaran G, Morency C, Roth B L, Toth B A, He P, Shuster L. SARof psilocybin analogs: discovery of a selective 5-HT 2C agonist. BioorgMed Chem Lett. 2005 15:4555-4559.Segman R H, Heresco-levy U, Finkel B, Inbar R, Neeman T, Schlafman M,Dorevitch A, Yakir A, Lerner A, Goltser T, Shelevoy A, Lerer B.Association between the serotonin 2C receptor gene and tardivedyskinesia in chronic schizophrenia: additive contribution of 5-HT2CSerand DRD3Gly alleles to susceptibility, Psychopharmacology 2000152:408-413.Setola V, Dukat M, Glennon R A, Roth B L. Molecular determinants for theinteraction of the valvulopathic anorexigen norfenfluramine with the5-HT_(2B) receptor. Mol Pharmacol 2005 68:20-33.Simansky K J. NIH symposium series: ingestive mechanisms in obesity,substance abuse and mental disorders. Physiology & Behavior 2005; 86:1-4.Siuciak J A, Chapin D S, McCarthy S A, Guanowsky V, Brown J, Chiang P,Marala R, Patterson T, Seymour P A, Swick A, Iredale P A. CP-809,101, aselective 5-HT2C agonist, shows activity in animal models ofantipsychotic activity. Neuropharmacology. 2007 52:279-290.Smith S R, Prosser W, Donahue D, Anderson C, Shanahan W. LorcaserinPhase 2b Clinical Study. American Diabetes Association, 2006.Stein T D, Anders N J, DeCarli C, Chan S L, Mattson M P, Johnson J A.Neutralization of transthyretin reverses the neuroprotective effects ofsecreted amyloid precursor protein (APP) in APP_(Sw) mice resulting intau phosphorylation and loss of hippocampal neurons: support for theamyloid hypothesis. J. Neurosci. 2004 24:7707-7717.Tecott L H, Sun L M, Akana S F, Strack A M, Lowenstein D H, Dallman M F,Julius D. Eating disorder and epilepsy in mice lacking 5-HT2c serotoninreceptors. Nature. 1995 374:542-546Tohda M, Takasu T, Nomura Y. Effects of antidepressants onserotonin-evoked current in Xenopus oocytes injected with rat brainmRNA. Eur J Pharmacol. 1989166:57-63.Veenstra-VanderWeele J, G. M. Anderson G M, Cook E H. Pharmacogeneticsand the serotonin system: initial studies and future directions. Eur. J.Pharmacol. 2000 410:65-181.Vickers S P, Dourish C T, and Kennett G A. Evidence that hypophagiainduced by D-fenfluramine and D-norfenfluramine in the rat is mediatedby 5-HT2C receptors. Neuropharmacology 2001 41: 200-209.

Vickers, S. P., Clifton, P. G., Dourish, C. T. and Tecott, L. H., 1999.Reduced satiating effect of d-fenfluramine in serotonin 5-HT2C receptormutant mice. Psychopharmacology 1999 143:309-314.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination of listed elements. The recitation of an element, or anembodiment herein includes that element or embodiment as any singleelement or embodiment or in combination with any other element,embodiments or portions thereof.

All references cited herein, whether in print, electronic, computerreadable storage media or other form, are expressly incorporated byreference in their entirety, including but not limited to, abstracts,articles, journals, publications, texts, treatises, technical datasheets, internet web sites, databases, patents, patent applications, andpatent publications.

Although the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations of theinvention may be devised by others skilled in the art without departingfrom the true spirit and scope of the invention. The claims are intendedto be construed to include all such embodiments and equivalentvariations.

1-8. (canceled)
 9. A method of inhibiting 5-HT2C in a subject identifiedas in need of such treatment, comprising administering a APT compound ofthe formula:

wherein, R₁ is independently 3-halophenyl, 3-haloalkylphenyl or3-alkoxyphenyl, wherein the 3-halophenyl, 3-haloalkylphenyl, or3-alkoxyphenyl are each optionally substituted with 1, 2, or 3independent halo, alkoxy, or CF₃; R₂ is H; or salt, hydrate or solvatethereof. 10-14. (canceled)
 15. A compound of the formula:

wherein, R₁ is independently 3-halophenyl, 3-haloalkylphenyl, or3-alkoxyphenyl, wherein the 3-halophenyl, 3-haloalkylphenyl, or3-alkoxyphenyl are each optionally substituted with 1, 2, or 3independent halo, alkoxy, or CF₃; and R₂ is H; or salt, hydrate orsolvate thereof.
 16. (canceled)
 17. A composition comprising a compoundof claim 15, or salt hydrate or solvate thereof, and a pharmaceuticallyacceptable carrier.
 18. A method of making a composition of claim 17comprising a compound of claim 15, or salt, hydrate or solvate thereof,and a pharmaceutically acceptable carrier.
 19. The compound of claim 15,or salt, hydrate or solvate thereof, wherein R₁ is 3-halophenyloptionally substituted with 1, 2, or 3 independent halo, alkoxy, or CF₃.20. The compound of claim 15, or salt, hydrate or solvate thereof,wherein R₁ is 3-haloalkylphenyl optionally substituted with 1, 2, or 3independent halo, alkoxy, or CF₃.
 21. The compound of claim 15, or salt,hydrate or solvate thereof, wherein R₁ is 3-alkoxyphenyl optionallysubstituted with 1, 2, or 3 independent halo, alkoxy, or CF₃.
 22. Thecompound of claim 15, or salt, hydrate or solvate thereof, wherein R₁ is3-halophenyl, 3-haloalkylphenyl, or 3-alkoxyphenyl.
 23. The compound ofclaim 15, or salt, hydrate or solvate thereof, wherein R₁ is3-halophenyl.
 24. The compound of claim 15, or salt, hydrate or solvatethereof, wherein R₁ is 3-haloalkylphenyl.
 25. The compound of claim 15,or salt, hydrate or solvate thereof, wherein R₁ is 3-alkoxyphenyl. 26.The compound of claim 15, or salt, hydrate or solvate thereof, whereinR₁ is 3-chlorophenyl.
 27. The compound of claim 15, or salt, hydrate orsolvate thereof, wherein R₁ is 3-CF₃-phenyl.
 28. The compound of claim15, or salt, hydrate or solvate thereof, wherein R₁ is 3-methoxyphenyl.29. The method of claim 9, wherein R₁ is 3-halophenyl.
 30. The method ofclaim 9, wherein R₁ is 3-haloalkylphenyl.
 31. The method of claim 9,wherein R₁ is 3-alkoxyphenyl.
 32. The method of claim 9, wherein R₁ is3-chlorophenyl.
 33. The method of claim 9, wherein R₁ is 3-CF₃-phenyl.34. The method of claim 9, wherein R₁ is 3-methoxyphenyl.